Method and composition for producing enhanced anti-inflammatory/ anti-catabolic and regenerative agents from autologous physiological fluid

ABSTRACT

Described are methods of producing an autologous composition useful for treatment of damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions and skin inflammatory disorders in a mammal. The method comprises preparing an anti-inflammatory/anti-catabolic component of the autologous composition comprising IL-1ra and TIMPs. An anti-inflammatory/anti-catabolic component is prepared comprising: collecting blood from the mammal; delivering the blood to a tube; incubating the blood at a temperature of from about 37° C. to about 39° C. for about 24 hours, preferably in the presence of sodium citrate; centrifuging the blood to separate the blood into a supernatant component and a cellular fraction; and collecting the supernatant component. The method further comprises preparing a regenerative component of the autologous composition comprising: collecting blood from the mammal; delivering the blood to a tube in the presence of about 4% citric acid; centrifuging the blood to separate a platelet-rich plasma component from a whole blood; collecting the platelet-rich plasma component; and mixing the supernatant component with the platelet-rich plasma component to provide the autologous composition. Also provided is a method of treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions and skin inflammatory disorders in a subject with the autologous composition, an autologous composition for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions and skin inflammatory disorders in a mammal and the use of the autologous composition for the treatment of damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions and skin inflammatory disorders in a mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. § 371 ofInternational Patent Application PCT/CA2015/000521, filed Sep. 30, 2015,designating the United States of America and published in English asInternational Patent Publication WO 2016/049746 A1 on Apr. 7, 2016,which claims the benefit under Article 8 of the Patent CooperationTreaty to Canadian Patent Application Serial No. 2900537 filed Aug. 13,2015, Canadian Patent Application Serial No. 2891445 filed May 14, 2015,and Canadian Patent Application Serial No. 2866480 filed Sep. 30, 2014.

TECHNICAL FIELD

This application is directed generally to medicine and, moreparticularly, to methods and compositions useful in, among other things,the treatment of damaged and/or injured connective tissues includingchronic tendinosis, chronic muscle tears (tendinitis), cartilage tears,chronic degenerative joint conditions such as osteoarthritis, as well aschronic inflammatory skin diseases including atopic dermatitis andchronic wounds.

BACKGROUND

Osteoarthritis (“OA”) is a degenerative joint disease characterized bycartilage damage and synovial inflammation. Previous data refer tochanges to a molecular inflammatory cascade that lead to a destructionof cartilage macromolecules and irreversible morphological changes.¹Considerable evidence has shown that IL-1, Tumor Necrosis Factor-alpha,IL-6,8 and metalloproteinases are predominant catabolic andpro-inflammatory molecules playing a major role in pathogenesis ofosteoarthritis.¹ These cytokines are produced by activated synoviocytes,mononuclear cells or by articular cartilage itself, and their cataboliceffect can be successfully blocked by inhibitory cytokines such asIL-4,10,13 and IL-1ra.¹

Similar inflammatory and catabolic pathways are involved in thepathogenesis of chronic tendonitis² and chronic muscle tear healingfailure.³ Tendon cells are subjected to continuous damage by producingincreased levels of IL-1,6, metalloproteinases (MMPs) and othercatabolic molecules.² Pro-inflammatory cytokines IL-1 and TNF-alpha areinvolved in pathogenesis of chronic myositis³ as well. Atopic dermatitis(eczema) is considered as the most common relapsing inflammatory skinconditions. Chronic wound (including diabetic wound) is a wound thatdoes not heal within three months due to poor circulation, neuropathy,immune disorders and complications of systemic illnesses, age, andrepeated trauma. All mentioned conditions are characterized bydisturbing cell signaling via cytokines and lost extracellular matrix(ECM) that forms the largest component of the dermal skin layer.Targeting special inflammatory and catabolic molecular pathways can havea beneficial therapeutic effect for inflammatory pathologies. Thiseffect could be achieved by using therapeutically active proteins.Presently, the pharmaceutical industry employs high-cost moleculargenetic technologies for recombinant protein production such as insulin,interferons, blood clotting factors, etc. However, these methods ofrecombinant protein generation include the expression of human genes ina bacterial cell. The patterns of post-translation protein modificationincluding glycosylation may be different than those naturally occurringin humans. This may result in instability of the product in the humanenvironment, decreasing of biological function or immune responseprovocation. Additionally, the cost of the final recombinant product isextremely high.

BRIEF SUMMARY

Described is a bioactive composition useful for treating damaged and/orinjured connective tissues, chronic tendinosis, chronic muscle tearsand/or chronic degenerative joint conditions such as osteoarthritis andskin inflammatory disorders. Also described is a method for making thecomposition. The composition includes an anti-inflammatorycomponent/anti-catabolic component. The anti-inflammatory component isalso an anti-catabolic component. For the purposes hereof, the terms“anti-inflammatory component” and “anti-catabolic component” can be usedinterchangeably. The composition may also include a regenerativecomponent that includes autologous platelet-rich plasma (PRP). Whereasmost PRP preparation protocols include an activation step resulting inthe immediate release of growth factors and cytokines from platelets,the present disclosure provides a use of a non-activated PRP componentfor future slow activation of injected composition by surroundingtissue.

The anti-inflammatory/anti-catabolic component preferably comprisesIL-1ra, an IL-1 receptor antagonist containing autologous serum. Inaddition, the anti-inflammatory/anti-catabolic component preferablycomprises an increased level of tissue inhibitors of metalloproteinases(TIMPs).

Provided is a method of producing an autologous composition for treatingdamaged and/or injured connective tissues, chronic tendinosis, chronicmuscle tears and/or chronic degenerative joint conditions and skininflammatory disorders in a mammal comprising the following steps:

-   -   A) preparing an anti-inflammatory/anti-catabolic component of        the autologous composition comprising TIMPs and IL-1ra, the step        of preparing the anti-inflammatory/anti-catabolic component        comprising the following steps: i) collecting an autologous        physiological fluid, preferably blood from the mammal; ii)        delivering the blood to a tube; iii) incubating the blood at a        temperature of from about 37° C. to about 39° C. for about 24        hours; iv) centrifuging the blood to separate the blood into a        supernatant component and a cellular fraction; and v) collecting        the supernatant component;    -   B) preparing a regenerative component of the autologous        composition comprising the following steps: i) collecting blood        from the mammal; ii) delivering the blood to a tube in the        presence of about 4% sodium citrate; iii) centrifuging the whole        blood to separate the platelet-rich plasma component; and iv)        collecting the platelet-rich plasma component; and    -   C) mixing the supernatant component of the        anti-inflammatory/anti-catabolic component with the        platelet-rich plasma component to provide the autologous        composition.

For the purposes of the instant disclosure, the terms “sodium citrate”and “citric acid” can be used interchangeably.

Also provided is a method of producing an autologous composition fortreating damaged and/or injured connective tissues, chronic tendinosis,chronic muscle tears and/or chronic degenerative joint conditions andskin inflammatory disorders in a mammal comprising the following steps:

-   -   A) preparing an anti-inflammatory/anti-catabolic component of        the autologous composition comprising TIMPs and IL-1ra, the step        of preparing the anti-inflammatory/anti-catabolic component        comprising the following steps: i) collecting blood from the        mammal; ii) delivering the blood to a tube including sodium        citrate; iii) incubating the blood at a temperature of from        about 37° C. to about 39° C. for about 24 hours; iv)        centrifuging the blood to separate the blood into a supernatant        component and a cellular fraction; and v) collecting the        supernatant component;    -   B) preparing a regenerative component of the autologous        composition comprising the following steps: i) collecting blood        from the mammal; ii) delivering the blood to a tube in the        presence of about 4% sodium citrate; iii) centrifuging the whole        blood to separate the platelet-rich plasma component; and iv)        collecting the platelet-rich plasma component; and    -   C) mixing the supernatant component of the        anti-inflammatory/anti-catabolic component with the        platelet-rich plasma component to provide the autologous        composition.

Further provided is a method of producing an autologous composition fortreating damaged and/or injured connective tissues, chronic tendinosis,chronic muscle tears and/or chronic degenerative joint conditions andskin inflammatory disorders in a mammal comprising the following steps:

-   -   i) collecting blood from the mammal;    -   ii) adding sodium citrate to a tube;    -   iii) delivering the blood to the tube;    -   iv) incubating the blood at a temperature of from about 37° C.        to about 39° C. for about 24 hours;    -   v) centrifuging the blood to separate the blood into a        supernatant component and a cellular fraction; and    -   vi) collecting the supernatant component.

Further provided is an autologous composition for treating damagedand/or injured connective tissues, chronic tendinosis, chronic muscletears and/or chronic degenerative joint conditions and skin inflammatorydisorders in a mammal produced by the method of the instant disclosure.

Also provided is an autologous composition for treating damaged and/orinjured connective tissues, chronic tendinosis, chronic muscle tearsand/or chronic degenerative joint conditions and skin inflammatorydisorders in a mammal, the composition comprising ananti-inflammatory/anti-catabolic component, preferably including sodiumcitrate, the anti-inflammatory/anti-catabolic component comprising TIMPsand IL-1ra. The composition further comprises a regenerative componentcomprising platelet-rich plasma.

Still further provided is an autologous composition for treating damagedand/or injured connective tissues, chronic tendinosis, chronic muscletears and/or chronic degenerative joint conditions and skin inflammatorydisorders in a mammal, the composition comprising ananti-inflammatory/anti-catabolic component, theanti-inflammatory/anti-catabolic component comprising a supernatantcomponent obtained from autologous blood of a mammal,anti-inflammatory/anti-catabolic component including IL-1ra and TIMPs,the composition further comprising a regenerative component comprisingplatelet-rich plasma obtained from the mammal. Theanti-inflammatory/anti-catabolic component preferably includes sodiumcitrate. Most preferably, the sodium citrate is a 4% solution of sodiumcitrate.

Also provided is the use of the autologous composition of thisdisclosure for the treatment of damaged and/or injured connectivetissues, chronic tendinosis, chronic muscle tears and/or chronicdegenerative joint conditions and skin inflammatory disorders in amammal.

Further provided is a method of treating a mammal for damaged and/orinjured connective tissues, chronic tendinosis, chronic muscle tearsand/or chronic degenerative joint conditions and skin inflammatorydisorders comprising the following steps:

-   -   (i) collecting blood from the mammal;    -   (ii) delivering the blood to a tube;    -   (iii) incubating the blood at a temperature of from about 37° C.        to about 39° C. for about 24 hours;    -   (iv) centrifuging the blood to separate the blood into a        supernatant component and a cellular fraction;    -   (v) collecting the supernatant component; and    -   (vi) preparing a regenerative component of the autologous        composition comprising the following steps:        -   collecting blood from the mammal;        -   delivering the blood to a tube in the presence of about 4%            citric acid;        -   centrifuging the blood to separate a platelet-rich plasma            component from a whole blood;        -   collecting the platelet-rich plasma component; and        -   mixing the supernatant component with the platelet-rich            plasma component to provide the autologous composition; and        -   administering the autologous composition to the mammal.

Further provided is a method of treating a mammal for damaged and/orinjured connective tissues, chronic tendinosis, chronic muscle tearsand/or chronic degenerative joint conditions and skin inflammatorydisorders comprising the following steps:

-   -   (i) collecting blood from the mammal;    -   (ii) adding sodium citrate to a tube;    -   (iii) delivering the blood to the tube;    -   (iv) incubating the blood at a temperature of from about 37° C.        to about 39° C. for about 24 hours;    -   (v) centrifuging the blood to separate the blood into a        supernatant component and a cellular fraction;    -   (vi) collecting the supernatant component; and    -   (vii) preparing a regenerative component of the autologous        composition comprising the following steps:        -   collecting blood from the mammal;        -   delivering the blood to a tube in the presence of citric            acid;        -   centrifuging the blood to separate a platelet-rich plasma            component from a whole blood;        -   collecting the platelet-rich plasma component;        -   mixing the supernatant component with the platelet-rich            plasma component to provide the autologous composition; and        -   administering the autologous composition to the mammal.

Further provided is a method of treating a mammal for damaged and/orinjured connective tissues, chronic tendinosis, chronic muscle tearsand/or chronic degenerative joint conditions and skin inflammatorydisorders comprising the following steps:

-   -   (i) collecting blood from the mammal;    -   (ii) delivering the blood to a tube in the presence of about 4%        sodium citrate;    -   (iii) incubating the blood at a temperature of from about 37° C.        to about 39° C. for about 24 hours;    -   (iv) centrifuging the blood to separate the blood into a        supernatant component and a cellular fraction;    -   (v) collecting the supernatant component; and    -   (vi) administering the supernatant component to the mammal.

The compositions and methods described herein are suitable forapplication to humans. They are also suitable for a wide range ofveterinary applications including the treatment of horses, dogs andcamels.

The instant disclosure provides an alternative product for treatingdegenerative joint disease in humans and for veterinary applicationsincluding horses, dogs and camels that is relatively safe effective,stable, regenerative, and cost effective.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of IL-1ra concentration in pg/ml versus time showing acomparison of the level of IL-1ra antagonist protein in human serumsamples with varying incubation conditions including stationary androcking at different time points.

FIG. 2 is a plot of IL-1ra concentration in pg/ml versus time showing acomparison of the level of IL-1ra antagonist protein in the human serumsamples at different time points in the presence of air, Ca⁺⁺ (inPhosphate Buffered Saline, PBS) and different concentration of serum.

FIGS. 3a and 3b are Doppler ultrasound images of a right paraspinal areaof a patient before treatment (FIG. 3a ) and normal post-treatmentcondition (FIG. 3b ).

FIGS. 4a and 4b are Doppler ultrasound images showing chronic Achillestendinosis characterized by excessive hyperemia (FIG. 4a , pre-treatmentstatus), that was resolved as a result of autologous compositiontreatment (FIG. 4b , post-treatment imaging).

FIG. 5 is a graph showing an average of concentration level of TIMP 1,TIMP 2 and TIMP 4 before and after a 24-hour incubation.

FIG. 6 is a graph showing an average baseline of knee joint painaccording to the Visual Analog Scale (“VAS”) scale in the eight patientsprior to and after injections.

FIG. 7 is a plot showing point values according to the WOMAC index foraverage levels of knee pain, stiffness and daily activity capabilitiesamong the eight patients.

FIG. 8 shows results in the form of a graph providing a breakdown of theaverage results after the first, second and third injections and two-and three-month follow-ups.

FIG. 9 is graph showing a comparison between the average concentrationlevel of platelet-derived growth factor (“PDGF”) in the human serumsamples before and after a 24-hour incubation.

FIG. 10 is a graph showing a comparison of the level of MMP2, MMP3,MMP7, MMP9 and MMP13 proteins in human serum samples before (baselinelevel) and after incubation at 37° C. for 24 hours.

FIG. 11 is a graph showing a comparison of the level of MMP2, MMP3,MMP7, MMP9 and MMP13 proteins in human serum samples before (baselinelevel) and after incubation at 37° C. for 24 hours in the presence ofsodium citrate.

FIG. 12 is a graph showing the MMP9 data from FIG. 11.

FIG. 13 is graph showing a comparison of the level of IL-1β in humanserum samples before (baseline level) and after incubation at 37° C. for24 hours, activated PRP, and in the final composition.

FIG. 14 is a graph showing a comparison of the level of TNF-α in humanserum samples before (baseline level) and after incubation at 37° C. for24 hours, activated PRP, and in the final composition.

FIG. 15 is a graph showing a comparison of the level of TIMP2 in thehuman serum samples before (baseline level) and after incubation at 37°C. for 24 hours, activated PRP, and in the final composition.

FIG. 16 is a graph showing a comparison of the level of IL-1ra in humanserum samples before (baseline level) and after incubation at 37° C. for24 hours, activated PRP, and in the final product.

FIG. 17 is a graph showing a comparison of the level of PDGF in thehuman serum samples before (baseline level) and after incubation in 37°C. for 24 hours, activated PRP, and in the final composition.

FIG. 18a is a plot showing point values according to the WOMAC index foraverage levels of pain among seventeen patients tested. Values areprovided for a baseline and 1 month post injections for CA− and CA+groups.

FIG. 18b is a plot showing point values according to the WOMAC index foraverage levels of stiffness among the seventeen patients tested. Valuesare provided for a baseline and 1 month post injections for CA− and CA+groups.

FIG. 18c is a plot showing point values according to the WOMAC index foraverage levels of daily activity capabilities among the seventeenpatients tested. Values are provided for a baseline and 1 month postinjections for CA− and CA+ groups.

FIG. 18d is a plot showing a statistical analysis of Visual Analog PainScale (VAS) among the seventeen patients tested. Values are provided fora baseline and 1 month post injections for CA− and CA+ groups.

FIG. 19a is a photograph of skin on the arm of a patient suffering frompsoriasis prior to treatment with the method of the present disclosure.

FIG. 19b is a photograph of skin on the arm of the patient sufferingfrom psoriasis three months following treatment with the method of thepresent disclosure.

DETAILED DESCRIPTION

The disclosure relates to a composition comprising an autologousanti-inflammatory/anti-catabolic component and preferably an autologousplatelet-rich plasma component with serum enriched by bioactive proteinshaving a synergistic anti-inflammatory/anti-catabolic, proliferative,tissue remodeling and regenerative effects.

Such a composition typically includes the following therapeuticallyactive proteins: IL-1ra⁶, IL-4⁷, IL-10^(8,9), IL-13¹⁰, PDGF¹¹,TGF-β^(10,11) and VEGF^(12,13,14,16).

IL-1ra is secreted by monocytes, adipocytes and epithelial cells.Therapeutically effective concentrations of this protein are achieved byincubating human monocytes at about 37° C. for about 24 hours.¹⁷IL-4,10,13, PDGF, TGF-β are the content of platelets and-granules andare delivered in the PRP component. IL-4,10,13 come from white bloodcells. PDGF is produced by platelets and TGF-B is released by plateletsand some T cells. Employing the synergistic effect of the mentionedproteins leads to generation of a potent bioactive autologous product.Thus, a combination of fresh-prepared PRP as a source of regenerativebiological factors and anti-inflammatory cytokines and growth factors,and the anti-inflammatory component comprising incubated autologousserum as a source of IL-1 inhibitor provides a powerful andcost-effective autologous therapeutic agent for treatment ofdegenerative conditions like osteoarthritis, chronic tendinosis andchronic muscle tears as well as skin inflammatory disorders.

As used herein, “treatment” includes palliative treatment, wherein painand/or inflammation is reduced in the subject.

It is surprising that the method of producing theanti-inflammatory/anti-catabolic component hereof leads to theproduction of an increased level of tissue inhibitors ofmetalloproteinases (TIMPs) in addition to the production of IL-1ra.Matrix metalloproteinases MMPs are believed to cause joint destructionwhen in an active state. TIMPs neutralize active MMPs, thereby providingan additional anti-catabolic benefit that is synergistic with IL-1ra.

It is further surprising that in producing theanti-inflammatory/anti-catabolic component hereof, the addition ofsodium citrate prior to incubating a patient's blood at a temperature offrom about 37° C. to about 39° C. for about 24 hours, prevents anincreased level of pathological molecular agents that have a cataboliceffect on joints such as MMP9, IL-1β and TNF-α, but yet does not lead toa significantly decreased level of anti-catabolic and regenerativeagents such as TIMPs, IL-1ra and PDGF.

The described method for producing an autologous composition for thetreatment of osteoarthritis, chronic tendinosis and chronic muscle tearas well as skin inflammatory disorders preferably comprises the step ofcollecting a mammal's autologous physiological fluid, preferably bloodby an aseptic technique. Preferably, the mammal is a human. However, thecompositions and methods hereof are also suitable for a wide range ofveterinary applications, for example, for the treatment of horses, dogsand camels.

The site of venipuncture and the surface of the collection tubes may becleaned with a 2 percent tincture of iodine solution. Before anycleansing of the site is begun, the patient may be asked about anyallergy to iodine. The tube covers are also cleaned with 70% alcoholsolution to avoid possible contamination before blood collection.

The composition is preferably prepared by culturing autologousphysiological fluid, preferably blood, at a temperature from about 37°C. to about 39° C. However, a person skilled in the art will appreciatethat the blood can be incubated at temperatures outside this range, forexample, from about 37° C. to about 40° C. with acceptable results. Mostpreferably, the temperature is between 37° C. and 38° C. The blood isincubated for about 24 hours for IL-1ra extracellular enrichment andpreferably for the production of TIMPs. Preferably, sodium citrate,preferably at a concentration of 4%, is added to a sterile glass tube ora polystyrene tube into which the blood is collected prior toincubation. In a particularly preferred embodiment, the incubation canbe in sterile glass tubes (Coviden) or polystyrene (BD) vacutainer tubeswith no additives. Further provided in an embodiment is the incubationof an autologous physiological fluid, preferably blood, on a rockerplatform (24 rpm) or in static conditions. Preferably, incubation iscarried out in static conditions as shown in FIG. 1.

Preferably, the incubation of blood is in the presence of 0.64-0.72 mMCa⁺⁺ to facilitate IL-1ra production.¹⁵ It is possible and advantageousin a particularly preferred embodiment to dilute incubated blood withsterile calcium chloride solution containing 0.64-0.72 mM Ca⁺⁺ in 9:1proportion by adding the solution using a sterile syringe and needledirectly to the tube with blood before the incubation (1 cc of thecalcium chloride solution to 9 cc whole blood) (FIG. 2). An equal partof sterile air may be added to the sterile tubes containing the blood toexpose the blood to atmospheric air for increasing IL-1ra production(FIG. 2). In a particularly preferred embodiment, the air will be passedthrough a 0.22 μm MILLEX® GP filter using a sterile syringe and needledirectly to the tube with the blood before the incubation.

Preferably, sodium citrate is added to the blood prior to incubation ina ratio of 9.5 parts of whole blood (9.5 cc):0.5 (0.5 cc) of preferably4% sodium citrate. The 4% citrate is preferably a 4% sodium citratesolution. Four percent sodium citrate solutions are readily commerciallyavailable.

The incubated blood is then subjected to centrifugation to separate thesupernatant component from the cellular fraction. The centrifugation iscarried out for about 10-20 minutes at about 4000-10000 rpm. Preferably,the centrifugation is carried out for 10 minutes at 4000 rpm.

The next step involves aspirating the supernatant and dividing it intoaliquots for future processing using a sterile technique. The procedureis carried out in a sterile environment (laminar flow hood with HEPAfilters). Three cc of the supernatant layer containing biologicallyactive agents are carefully drawn by sterile syringe and needle.Prolonged storage of IL-1ra containing product is accomplished byfreezing aliquots at about −20° C. and storing for up to 6 months or upto one year at about −70° C.

The preparation of the regenerative component comprising PRP theninvolves drawing blood into vacutainer tubes. This is preferably carriedout in the presence of 4% citric acid. Preferably, in 9.5 parts of wholeblood (9.5 cc): 0.5 (0.5 cc) of 4% citric acid ratio. The blood is thensubjected to centrifugation, preferably for about 30 seconds, at about7500 rpm to isolate the PRP fraction. The centrifugation parameters areused in preferred embodiments for the PRP preparation as a part of thefinal product for the osteoarthritis and chronic tendinosis treatmentand skin disorders. The PRP fraction is drawn by a sterile syringe andneedle under sterile conditions. In a particularly preferred embodimentfor the treatment of chronic tear, a leukocyte buffy coat fraction isadded to the PRP as an additional VEGF source in order to promote newblood vessel development in the affected site. The buffy coat layer andplasma is collected manually by sterile syringe and needle after wholeblood centrifugation as set out above or using a commercially availableHarvest SMARTPREP® system. The regenerative component comprising PRP isnot subject to freezing or other storage. The autologous composition isto be administered to a patient promptly after mixing the regenerativecomponent with the anti-inflammatory/anti-catabolic component.

The anti-inflammatory/anti-catabolic component comprising IL-1ra andpreferably TIMPs containing blood is meted with the regenerativecomponent comprising PRP fraction, preferably in a 1:1 ratio, to obtainthe final product.

The product is injected for the future slow activation by tendon-derivedcollagen¹⁸ and tissue-derived thrombin.

The disclosure is further described with the aid of the followingillustrative Examples.

EXAMPLES Example 1—Comparison of Extracellular IL-1ra Production UponDifferent Culture Conditions

As graphically shown in FIG. 1, a comparison was carried out of thelevel of IL-1ra antagonist protein in the human serum samples that wereexposed to incubation conditions at different time points includingstationary versus rocking incubation. IL-1ra is secreted by theactivated blood monocyte, macrophages. Such activation is achieved bycontact between blood cells and the internal surfaces of the collectiontubes through the use of an agitation process. By increasing theinternal surface area exposed to the cellular component, the cellactivation process and bioactive molecule secretion can be maximized.

Peripheral blood from ten healthy male and female volunteer donors (21to 60 years old) was collected by venipuncture under sterile conditionsto sterile 10 ml glass tubes. One tube was manipulated according to astandard procedure with no incubation step (control sample). Sampleswere incubated for 3.5 hours, 7 hours, and 24 hours at 37° C. with andwithout agitation on a rocker platform (24 rpm). Incubated samples werecentrifuged for 10 minutes at 4,000 rpm and then filtered, and the finalconcentrations of IL-1ra were compared to those of unprocessed controlsamples (0 hours) according to manufacture protocol (available on theinternet at bio-rad.com/webroot/web/pdf/lsr/literature/10014905.pdf). Aone-way ANOVA test revealed a significant increase of IL-1raconcentration in the serum after 24 hours of incubation only; nosignificant IL-1ra concentration increase was observed in 3.5-hour and7-hour incubated samples. Additionally, no significant difference wasobserved between stationary and rocking incubation conditions. IL-1raconcentration was evaluated using MAGPIX® LUMINEX® technology.

FIG. 2 shows a comparison of the level of antagonist protein in thehuman serum samples that were exposed to the following incubationconditions: 24 hours incubation in the presence of air, Ca⁺⁺ (inPhosphate Buffered Saline, PBS) and different concentrations of serum. Aone-way ANOVA test revealed a significant increase of IL-1ra productionin the serum after 24 hours of incubation upon all mentioned conditionsbesides culturing in 50% diluted blood.

Example 2—Case Reports of Patients Diagnosed with Osteoarthritis andChronic Tendinosis and Treated with the Autologous Composition

Methods and Materials:

For each patient herein, the anti-inflammatory/anti-catabolic componentof the autologous composition comprising IL-1ra and TIMPs was preparedby: collecting blood from the patient; delivering the blood to a tube;incubating the blood at a temperature of from about 37° C. to about 39°C. for about 24 hours; centrifuging the blood to separate the blood intoa supernatant component and a cellular fraction; and collecting thesupernatant component of the anti-inflammatory component. Likewise, theregenerative component of the autologous composition was prepared by:collecting blood from the patient; delivering the blood to a tube in thepresence of about 4% citric acid; centrifuging the blood to separate aplatelet-rich plasma component from a whole blood component; collectingthe platelet-rich plasma component; and mixing the supernatant componentof the anti-inflammatory/anti-catabolic component with the platelet-richplasma component to provide the autologous composition. The autologouscomposition was then administered to the patient.

Case 1: S, 61 Years Old.

Diagnosis: The patient reported bilateral insidious onset knee pain,which began a few years prior and which had increased within thepreceding 6 months. MRI of the knees showed severe OA of the knees:severe chondrosis of the medial compartment with increased amount offull-thickness cartilage loss involving the right and a femoral condylein the right knee and full thickness chondral loss involving theposterior aspect of the medial femoral trochlea with underlying edema inthe right knee. One year earlier, the patient had a Cortisone injectionIA, which provided one month relief. Physical exam: Knees Range ofMotion (ROM) was full, all ligaments were normal, small bilateraleffusion neurovascular exam was normal. VAS was 60.

Treatment: Bilateral injections of the local autologous composition forthe patient into the patient's knees three times, a week apart.

Results: After the first injection bilaterally of the local autologouscomposition for the patient, the patient reported significantimprovement, VAS was 3. At the time of the third injection, ROM wasfull, all ligaments were normal, with no joint line pain, and noeffusion. The patient reported strong pain reduction, VAS was 10, thepatient went back to physical activity. Three months later, a follow-upexam showed that the patient was pain free.

Case 2: E, 64 Years Old.

Diagnosis: Active male presented with VAS 60 in the left hip. Pain withdaily activities and significant impairments with walking for a longdistance. Mill showed mild OA in both hips: bilateral hip jointdegeneration and acetabular labral degenerative tearing. Physiotherapytreatment had limited success in terms of pain relief.

Treatment: Ultrasound-guided local autologous composition injectionprepared for the patient into left hip ×2, a week apart.

Results: After first injection of the local autologous composition, thepatient reported an 85% improvement in pain (patient personalassessment). After second injection of the local autologous composition,the VAS was 10. Five months later VAS was also 10.

Case 3: A, 70 Years Old.

Diagnosis: A female patient presented with chronic pain (VAS was 6)tenderness and swelling in the left knee. She had much difficulty inwalking, standing, and climbing stairs. She had been to a physicaltherapist (6 visits), a chiropractor (6 visits) for help with her kneepain with no results. Mill showed severe OA in left knee, with narrowingof the medial compartment due to full-thickness cartilage loss in theweight-bearing portion of the medial femoral condyle and medial tibialplateau.

Treatment: Local autologous composition was made and injections weremade into left knee three times, a week apart.

Results: Five-month follow-up visit after treatment with the localautologous composition: the patient reported about 80% improvement insymptoms, no swelling; VAS is 20.

Case 4: J, 26 Years Old, Professional Swimmer.

Diagnosis: Status post paraspinal muscle injury, chronic paraspinaltendonitis. Patient complained of pain over the paraspinal muscle.Prolonged physiotherapy had shown no results. Back ROM is full,neurovascular exam is normal. Area of right paraspinal hyperemia isidentified on color Doppler FIG. 3a , VAS was 80.

Treatment: Inflamed area was marked using the ultrasound technique,autologous composition was injected intramuscularly ×4, a week apart.

FIGS. 3a and 3b are Doppler ultrasound images that revealed a hyperemiain the right paraspinal area before treatment (FIG. 3a ) and normalpost-treatment condition (FIG. 3b ).

Results: Doppler showed no inflammation (FIG. 3b ), VAS was 10 fourmonths after the therapy.

Case 5: S, 18 Years Old.

Diagnosis: Status post right Achilles tear. The patient complained ofpain in the Achilles insertion: chronic Achilles tendonitis andtenosynovitis. Doppler showed severe intra-tendon hyperemia in the area(FIG. 4a ). VAS was 60.

Treatment: Prolonged physiotherapy and chiropractic treatment did notreveal any positive result. Autologous composition was prepared andinjected into the tendon three times, a week apart, utilizing ultrasoundguidance.

FIGS. 4a and 4b are Doppler ultrasound images that showed chronicAchilles tendinosis characterized by excessive hyperemia (FIG. 4a ,pre-treatment status), that was resolved as a result of autologouscomposition treatment (FIG. 4b , post-treatment imaging).

Results: no hyperemia displayed on Doppler imaging (FIG. 4b ), VAS was 0at five months post-injection follow-up assessment.

Example 3—In Vitro Studies

Peripheral blood from healthy male and female volunteer donors (21 to 60years old) was collected by venipuncture under sterile conditions tosterile 10 ml glass tubes. One tube was manipulated according to astandard procedure with no incubation step (control sample). Sampleswere exposed to different incubation conditions and 500 μl of serum fromeach sample was used for an assay. Samples were centrifuged at 10,000×gfor 10 minutes at 4° C. prior to analysis to remove cell debris andaggregates. BioPlex Pro™ Human Cytokine 27-plex Panel, Human TIMPMagnetic LUMINEX® Performance Assay 4-plex Panel™ (Bio-Rad Laboratories,Canada, LTD) analysis with MAGPLEX® beads was performed in a flat-bottommicrotiter plate according to the manufacturer's instructions (availableon the internet atbio-rad.com/webroot/web/pdf/lsr/literature/10014905.pdf).

Briefly, samples were diluted 1:4 in sample diluent. Standard wasreconstituted and diluted in a four-fold dilution series.Antibody-coupled capture beads were prepared and plated. The beadsolution was vortexed before addition to each well. The plate waswashed, and all wash steps were performed manually. First, the washsolution was added to the plate that was subsequently covered withsealing tape. The plate was incubated on a shaker for 30 seconds at 1100rpm and then for 1.5 minutes at 300 rpm. The plate was taken off theshaker and was incubated on a magnet for 1 minute before the supernatantwas discarded. After washing, diluted samples and standards were addedin duplicates to the beads in the wells. The plate was incubated on ashaker for 30 minutes and after incubation and washing, detectionantibodies were added for 30 minutes to each well. The plate was againincubated on a shaker and after another washing step, streptavidinsolution was added for 10 minutes to the wells. After a last incubationstep, the beads were re-suspended in assay buffer and the plate was readwith a MAGPIX® (Luminex Corporation) using the xPONENT® software(Luminex Corporation, Austin, Tex., USA). The results were analyzedusing the xPONENT® software. The absolute concentrations of the sampleswere determined by the construction of a standard curve for eachanalyte.

Statistical analysis: All statistical tests were performed usingGRAPHPAD PRISM® version 5.01. Statistical comparisons were performedusing analyses of variance (ANOVA) with Bonferroni post-test tests forcomparisons between groups. The number of analyzed experiments was ≥3,and data were shown as mean±s.e.m., p<0.05.

FIG. 5 is a graph showing a comparison of the level of TIMP 1, TIMP 2and TIMP 4 (MMPS antagonists) proteins in the human serum samples before(baseline level) and after incubation at 37° C. for 24 hours. A two-wayANOVA test revealed a statistically significant increase in the levelsof TIMP 1 and TIMP 2 at 24 hours post incubation.

FIG. 9 is a graph showing a comparison between the average concentrationlevel of PDGF in the unprocessed human serum samples and an averagelevel of PDGF in 24-hour-incubated samples. Test data analysis showed astatistically significant increase of PDGF protein concentration inprocessed samples. PDGF concentration was evaluated using MAGPIX®LUMINEX® technology.

Example 4—Results from Patients Treated for Knee Osteoarthritis JointPain

Eight patients were treated for symptoms of knee osteoarthritis jointpain. The patients were provided with four injections of the localautologous composition, according to the procedure summarized in Example2, one week apart.

The patients were assessed after receiving the injections, with VisualAnalog Pain Scale (VAS) and the Western Ontario and McMasterUniversities Arthritis Index (WOMAC) questionnaire for assessing pain,stiffness, and physical function in patients with hip and/or kneeosteoarthritis. The analysis of the WOMAC questionnaire data showed astatistically significant improvement in the patient's daily activitiesand a statistically insignificant but positive dynamic of improvement inpain and stiffness parameters for the prolonged period (up to 3 months)as shown in FIG. 7. A statistical analysis of the Visual Analog PainScale revealed a significant pain reduction after the third injection asshown in FIG. 6.

FIG. 6 is a bar graph showing comparison of an average baseline and postinjections of joint pain according to the VAS scale in eight patients.The results show a statistically significant average decrease in painwith stable effect up to 3 months.

FIG. 7 is a plot showing point values according to the WOMAC index foraverage levels of pain, stiffness and daily activity capabilities amongthe eight patients. Values are provided for a baseline, afterinjections, two months and three months post injections.

FIG. 8 shows results in the form of a graph among the eight patientsproviding a breakdown of the average results after the first, second,third injections, two months and three months post the fourth injection.

The results shown in FIGS. 6-8 are average results among the eightpatients tested.

Example 5—Effect of Addition of Sodium Citrate (CA) Prior to Incubationin Preparation of Anti-Inflammatory/Anti-Catabolic Component

Fifteen patients were tested to determine the effect of the addition ofsodium citrate (CA) to the patient's blood, prior to incubation of theblood for about 24 hours, on levels of MMPs including MMP9, IL-1β,TNF-α, TIMP2, Il-1ra, and PDGF in the anti-inflammatory component and inthe final product being the combination of the anti-inflammatorycomponent and the regenerative or PRP component.

The terms “blood” and “serum” are used interchangeably in the followingdiscussion.

Methods and Materials:

For each patient herein, an anti-inflammatory/anti-catabolic componentwas prepared as follows. About 9.5 cc of blood was collected from thepatient. The about 9.5 cc of blood was then delivered to a first tubecontaining about 0.5 cc of saline solution. The saline solutioncomprised about 0.9% NaCl. Another about 9.5 cc of blood was collectedfrom the patient and delivered to a second tube containing about 0.5 ccof 4% sodium citrate. After collecting the blood, the first and secondtubes were incubated at a temperature of from about 37° C. to about 39°C. for about 24 hours. The first and second tubes were then centrifugedto separate the blood in each tube into a supernatant component and acellular fraction. The supernatant components of the first and secondtubes were collected separately to provide a firstanti-inflammatory/anti-catabolic component emanating from the first tubecontaining saline and a second anti-inflammatory/anti-cataboliccomponent emanating from the second tube containing sodium citrate. Foreach patient, a regenerative component was prepared by: collecting bloodfrom the patient; delivering the blood to a tube in the presence ofabout 4% citric acid; and centrifuging the blood to separate aplatelet-rich plasma component from a whole blood component. For eachpatient, a platelet-rich plasma component was collected and mixed withthe supernatant component of the first and second tubes, respectively,to provide a first final product emanating from the first tubecontaining saline and a second final product emanating from the secondtube containing sodium citrate.

For each patient, a baseline level of MMP2, MMP3, MMP7, MMP9, MMP13,IL-1β, TNF-α, TIMP2, Il-1ra, and PDGF was measured immediately afterdrawing the blood. For each of the fifteen patients, a level of MMP9,IL-1β, TNF-α, TIMP2, Il-1ra, and PDGF was measured in the first tubecontaining saline and the patient's blood about 24 hours afterincubation. Similarly, for each of the fifteen patients, a level ofMMP9, IL-1β, TNF-α, TIMP2, Il-1ra, and PDGF was measured in the secondtube containing sodium citrate and the patient's blood about 24 hoursafter incubation. For each patient, the platelet-rich plasma componentwas mixed with thrombin and then the levels of MMP9, IL-1β, TNF-α,TIMP2, Il-1ra, and PDGF were measured. For each patient, a level ofMMP9, IL-1β, TNF-α, TIMP2, Il-1ra, and PDGF was measured in each of thefirst and second final products. The measurements shown graphically inFIGS. 10-17 represent averages of the fifteen patients tested.

In the fifteen patients tested, an average level of IL-1ra in nativeserum (blood) at 0 hours of incubation was measured to be 9±4 pg/ml. Inthe final product resulting from the combination of theanti-inflammatory/anti-catabolic component with the regenerative or PRPcomponent, the average measurement for IL-1ra in native serum (blood)was 920±80 pg/ml. This was the case, both with the addition of sodiumcitrate to the anti-inflammatory/anti-catabolic component and where nosodium citrate was added.

In the fifteen patients tested, an average level of PDGF in native serum(blood) at 0 hours of incubation was measured to be 1100±300 pg/ml. Inthe final product resulting from the combination of theanti-inflammatory/anti-catabolic component with the regenerative or PRPcomponent, the average measurement for PDGF in native serum (blood) was1920±380 pg/ml. This was the case, both with the addition of sodiumcitrate to the anti-inflammatory/anti-catabolic component and where nosodium citrate was added.

In the fifteen patients tested, an average level of TIMP2 in nativeserum (blood) at 0 hours of incubation was measured to be 1800±150pg/ml. In the final product resulting from the combination of theanti-inflammatory/anti-catabolic component with the regenerative or PRPcomponent, the average measurement for TIMP2 in native serum (blood) was5500±360 pg/ml. This was the case, both with the addition of sodiumcitrate to the anti-inflammatory/anti-catabolic component and where nosodium citrate was added.

In these figures, serum 0 hours is the baseline measurement of the levelof the analyte in the patients' blood measured immediately uponcollection. Serum 24 hours+0.5 cc saline is the level of the analyte inthe patients' blood mixed with saline solution after 24 hours ofincubation. Serum 24 h+0.5 cc CA is the level of the analyte in thepatients' blood mixed with 4% sodium citrate solution after 24 hours ofincubation. PRP+Thrombin is the level of the analyte in the PRPcomponent combined with thrombin as a clotting agent. This measurementshows a baseline level of analyte in the PRP component.PRP+Serum+Thrombin or PRP+Serum 24 h+Thrombin is the level of theanalyte in the final product resulting from the combination of theanti-inflammatory/anti-catabolic component with the regenerative or PRPcomponent where the anti-inflammatory/anti-catabolic component wasprepared with saline only and no sodium citrate. PRP+Serum+CA+Thrombinor PRP+Serum 24 h+CA+Thrombin is the level of the analyte in the finalproduct resulting from the combination of theanti-inflammatory/anti-catabolic component with the regenerative or PRPcomponent where the anti-inflammatory/anti-catabolic component wasprepared with sodium citrate.

In preparing the anti-inflammatory/anti-catabolic component as set outin the procedure of Example 2, it was observed that the production ofthis component results in an increased level of IL-1ra(anti-inflammatory agent), TIMPs (anti-catabolic agents) and PDGF(regenerative agent) after the incubation step. The method of producingthe autologous bioactive composition is based on the ability ofactivated blood mononuclear cells to secrete positive bioactivemolecules. However, it has now been determined that the same immunecells un-selectively produce both pathological molecular agents andtheir inhibitors upon their activation. Thus, the autologous bioactivecomposition also contains an elevated concentration of catabolicmolecules such as MMP9. This was confirmed by quantitative measurementof four MMP types in the anti-catabolic component prepared afterincubating the patients' blood in the presence of saline at about 37° C.to about 39° C. for about 24 hours, as shown in FIG. 10. While theconcentrations of MMP 2,3,7,13 were not found significantly changedafter incubation, the concentration of MMP9, which is most critical forosteoarthritis pathogenesis, was significantly increased, as shown inFIG. 10. A one-way ANOVA test revealed a statistically significantincrease in the levels of MMP9 at 24 hours post incubation.

The presence of increased MMP9 concentration in the autologous bioactivecomposition may result in an adverse effect manifestation during thepatient treatment process. It is, therefore, desired to selectivelyeliminate this negative component. The presence of the anticoagulant,sodium citrate, significantly down-regulates MMP9 release by human bloodleukocytes.¹⁹ Human blood in the presence of sodium citrate (CA) at aratio of 9.5 cc blood:0.5 cc sodium citrate was incubated at atemperature of from about 37° C. to about 39° C. for 24 hours and foundstrong dynamic changes, especially in MMP9 secretion as shown in FIGS.11 and 12. Particularly, adding sodium citrate in preparing theanti-inflammatory/anti-catabolic component significantly decreased MMP9concentration in the final product of the combination of theanti-inflammatory/anti-catabolic component and the regenerativecomponent (thrombin-activated PRP+24 h serum). The MMP9 level after a24-hour incubation did not change compared to the baseline level (0hours) where sodium citrate was added in preparing theanti-inflammatory/anti-catabolic component. A measurement of the levelof MMP9 in PRP component with thrombin was made to show a baseline levelof MMP9 in the PRP component. This measurement shows that no significantquantity of MMP9 is produced in generating the PRP component.

Given that the blood incubation process leads to an elevation of thepro-catabolic molecule MMP9, concentrations of other pathologicalmolecular agents, namely IL-1β and TNF-α were tested in incubated serumand in the final product of the combination of theanti-inflammatory/anti-catabolic component and the regenerativecomponent (thrombin-activated PRP+blood serum incubated for 24 hours).The effect of sodium citrate on these molecules released by activatedleukocytes during incubation was tested. It was found that although bothIL-1β and TNF-α concentrations are significantly increased uponincubation. This effect could be efficiently blocked by adding sodiumcitrate in preparing the anti-inflammatory/anti-catabolic component, asshown in FIGS. 13 and 14.

With reference to FIG. 13, a two-way ANOVA test revealed a statisticallysignificant decrease in IL-10 concentration in 24-hour-incubated bloodserum from about 37° C. to about 39° C. in the presence of sodiumcitrate as well as in the final product (PRP+serum 24 h+sodium citrate(CA)+Thrombin) as compared to the previous method for productpreparation (PRP+serum 24 h+saline). As referred to throughout, thefinal product (PRP+serum 24 h+CA+Thrombin), is the combination of theanti-inflammatory/anti-catabolic component and the regenerativecomponent.

With reference to FIG. 14, a two-way ANOVA test revealed a statisticallysignificant decrease of TNF-α concentration in 24-hour-incubated serumfrom about 37° C. to about 39° C. in the presence of sodium citrate aswell as in the final product (PRP+serum 24 h+CA+Thrombin) as compared tothe previous method of product preparation (PRP+serum 24 h+saline).

To evaluate whether the addition of sodium citrate to theanti-inflammatory/anti-catabolic component affects pathologicalmolecular agent inhibitors during bioactive composition preparation,TIMPs, Il-1ra and PDGF concentrations were tested upon similarconditions. No negative effects of sodium citrate on TIMPs, Il-1ra andPDGF production were observed as shown in FIGS. 15, 16, and 17.

With reference to FIG. 15, a comparison of the level of TIMP2 in thehuman serum samples was conducted before (baseline level), afterincubation at 37° C. for 24 hours, activated PRP and in the finalproduct. A two-way ANOVA test revealed that sodium citrate does notdecrease the concentration of TIMPs in 24-hour-incubated serum in thepresence of sodium citrate as well as in the final product (PRP+serum 24h+CA+Thrombin) as compared to the previous method of product preparation(PRP+serum 24 h+saline).

With reference to FIG. 16, a two-way ANOVA test revealed that sodiumcitrate does not decrease IL-1ra concentration in 24-hour-incubatedserum in the presence of sodium citrate as well as in the final product(PRP+serum 24 h+CA+Thrombin) as compared to the previous method ofproduct preparation (PRP+serum 24 h+saline).

With reference to FIG. 17, a two-way ANOVA test revealed that sodiumcitrate does not decrease PDGF concentration in 24-hour-incubated serumas well as in the final product (PRP+serum 24 h+CA+Thrombin) as comparedto previous method product preparation (PRP+serum 24 h+saline).

Example 6—Treatment of Patients with Symptoms of Knee OsteoarthritisJoint Pain

Seventeen patients were treated for symptoms of knee osteoarthritisjoint pain according to the method described herein, as summarized inExample 2. Two groups of patients were provided with two injections oneweek apart. A first group of seven patients received a product includingthe anti-inflammatory/anti-catabolic component prepared with no presenceof sodium citrate (CA−). A second group of ten patients received aproduct including the anti-inflammatory/anti-catabolic componentprepared with the presence of sodium citrate (CA+). Both treatments didnot result in any adverse events and were found to be safe andeffective.

The patients were assessed with the Western Ontario and McMasterUniversities Arthritis Index (WOMAC) questionnaire for assessing pain,stiffness, and physical function in patients with hip and/or kneeosteoarthritis. A one month post-injection preliminary analysis of theWOMAC questionnaire data showed a statistically significant improvementin the patients' pain, stiffness and daily activities in patientstreated with the anti-inflammatory/anti-catabolic component havingcitrate (CA+) as shown in FIGS. 18a, 18b, and 18c , respectively. Withthe group of patients treated with the anti-inflammatory/anti-cataboliccomponent not having citrate (CA−), a statistically insignificant butpositive dynamic of improvement in the mentioned parameters was observedas shown in FIGS. 18a, 18b, and 18c , respectively. A statisticalanalysis of Visual Analog Pain Scale (VAS) revealed a significant painreduction in both groups, as shown in FIG. 18 d.

Example 7—Chronic Inflammatory Skin Diseases

The method of Example 2 was performed on a female patient aged 59suffering from severe psoriasis, over a four week period. The autologouscomposition produced by combining the anti-inflammatory/anti-cataboliccomponent with the platelet-rich plasma component was administered tothe female patient in four separate injections of the autologouscomposition one week apart. The treatments involved intra-dermalinjection of the entire lesion site. Dramatic visual changes wereobserved by four weeks post treatment. The outcome was stable afterthree months post injection with the effects of psoriasis beingeradicated. FIG. 19a is a photograph of the affected area of the femalepatient's arm prior to treatment. FIG. 19b is a photograph of theaffected area of the female patient's arm three months after the fourthinjection.

The methods of Example 2 are performed with and without the addition ofsodium citrate in preparing the anti-inflammatory/anti-cataboliccomponent on subjects suffering from chronic inflammatory skin diseasessuch as atopic dermatitis and chronic wounds. The chronic inflammatoryskin diseases reduce in severity.

Example 8—Horses, Dogs and Camels

The methods of Example 2 with and without the addition of sodium citratein preparing the anti-inflammatory/anti-catabolic component areperformed on horses, dogs and camels suffering from damaged and/orinjured connective tissues. The pain associated with the damaged and/orinjured connective tissues reduces in severity.

Although the invention has been described with reference to illustrativeembodiments, it is to be understood that the invention is not limited tothese precise embodiments. Numerous modifications, variations, andadaptations may be made to the particular embodiments of the inventiondescribed above without departing from the scope of the invention. Thescope of the claims should not be limited by the preferred embodimentsset forth in the examples, but should be given the broadestinterpretation consistent with the description as a whole.

REFERENCES

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The content of these references is hereby incorporated by reference.

The invention claimed is:
 1. A method of treating a mammal sufferingfrom damaged and/or injured connective tissues, chronic tendinosis,chronic muscle tears and/or chronic degenerative joint conditions andskin inflammatory disorders, the method comprising the following steps:preparing an anti-inflammatory/anti-catabolic component of an autologouscomposition comprising IL-1ra, tissue inhibitor of matrixmetalloproteinase 1 (“TIMP 1”), and tissue inhibitor of matrixmetalloproteinase 2 (“TIMP 2”), the step of preparing theanti-inflammatory/anti-catabolic component comprising the followingsteps: collecting blood from the mammal; delivering the blood to a tube;incubating the blood at a temperature of from about 37° C. to about 39°C. for about 24 hours, so as to increase the concentration of TIMP 1therein to greater than 6,500 pg/ml and to increase the concentration ofTIMP 2 therein to greater than 4,000 pg/ml; centrifuging the blood toseparate the blood into a supernatant component and a cellular fraction;and collecting the supernatant component of theanti-inflammatory/anti-catabolic component; preparing a regenerativecomponent of the autologous composition comprising the following steps:collecting blood from the mammal; delivering the blood to a tube in thepresence of about 4% citric acid; centrifuging the blood to separate aplatelet rich plasma component from a whole blood; and collecting theplatelet rich plasma component; mixing the supernatant component withthe platelet rich plasma component to provide the autologouscomposition; and administering the autologous composition to the mammal.2. The method according to claim 1, wherein the tube for receiving theblood in the preparation of the anti-inflammatory/anti-cataboliccomponent is a vacutainer tube constructed of glass.
 3. The methodaccording to claim 1, wherein the tube for receiving the blood in thepreparation of the anti-inflammatory/anti-catabolic component is avacutainer tube constructed of polystyrene.
 4. The method according toclaim 1, wherein the step of preparing ananti-inflammatory/anti-catabolic component of the autologous compositioncomprising IL-1ra and TIMPs further comprises the step of culturing theblood in the presence of Ca⁺⁺ to facilitate IL-1ra production.
 5. Themethod according to claim 4, wherein the blood is cultured with asterile calcium chloride solution comprising about 0.64 to about 0.72 mMCa⁺⁺ in a 9:1 proportion by adding the solution using a sterile syringeand needle directly to the tube with autologous physiological fluidbefore the incubation step.
 6. The method of claim 4, further comprisingthe step of adding sterile air to the tube comprising the blood forincreasing IL-1ra production.
 7. The method according to claim 1,wherein the supernatant component is divided into aliquots and storedfor future use.
 8. The method according to claim 1, wherein thesupernatant component is divided into aliquots and frozen for futureuse.
 9. The method according to claim 1, wherein the step of deliveringthe blood to a tube including a quantity of about 4% citric acidcomprises providing a ratio of 9.5 parts of whole blood: 0.5 parts of 4%citric acid.
 10. The method according to claim 1, wherein a leukocytebuffy coat fraction is added to regenerative component as an additionalVEGF source.
 11. The method according to claim 1, wherein the step ofmixing the supernatant component with the platelet rich plasma componentto provide the autologous composition comprises mixing the supernatantcomponent with the platelet rich plasma component in a 1:1 ratio. 12.The method according to claim 1, wherein the mammal is a human.
 13. Amethod of treating damaged and/or injured connective tissues, chronictendinosis, chronic muscle tears and/or chronic degenerative jointconditions and skin inflammatory disorders in a mammal, the methodcomprising the following steps: preparing ananti-inflammatory/anti-catabolic component of an autologous compositioncomprising IL-1ra and tissue inhibitors of matrix metalloproteinase(“TIMPs”), the step of preparing the anti-inflammatory/anti-cataboliccomponent comprising the following steps: collecting blood from themammal; adding an about 4% sodium citrate solution to a tube; deliveringthe blood to the tube; incubating the blood at a temperature of fromabout 37° C. to about 39° C. for about 24 hours; centrifuging the bloodto separate the blood into a supernatant component and a cellularfraction; and collecting the supernatant component of theanti-inflammatory/anti-catabolic component; preparing a regenerativecomponent of the autologous composition comprising the following steps:collecting blood from the mammal; delivering the blood to a tubeincluding a quantity of about 4% citric acid solution; centrifuging theblood to separate a platelet rich plasma component from the blood;collecting the platelet rich plasma component; mixing the supernatantcomponent with the platelet rich plasma component to provide theautologous composition; and administering the autologous composition tothe mammal, wherein preparing the anti-inflammatory/anti-cataboliccomponent with the about 4% sodium citrate solution significantlydecreases MMP9 concentration in the anti-inflammatory/anti-cataboliccomponent and in the autologous composition in comparison to anautologous composition prepared without utilizing the about 4% sodiumcitrate solution to prepare the anti-inflammatory/anti-cataboliccomponent.
 14. The method of claim 13 wherein the 4% sodium citratesolution is added to the tube to provide a ratio of about 0.5:9.5 of the4% sodium citrate solution to blood upon delivery of the blood to thetube.
 15. A method of treating a mammal having damaged and/or injuredconnective tissues, chronic tendinosis, chronic muscle tears and/orchronic degenerative joint conditions and skin inflammatory disorders,the method comprising: preparing a first component of an autologouscomposition by: admixing blood from the mammal with an amount of about4% sodium citrate solution to form an admixture, incubating theadmixture at a temperature of from about 37° C. to about 39° C. forabout 24 hours, wherein the sodium citrate significantly decreases MMP9concentration in the admixture in comparison to such an admixturewithout sodium citrate therein; centrifuging the thus incubatedadmixture to separate the admixture into a supernatant component and acellular fraction; and collecting the supernatant component as the firstcomponent; preparing a second component of the autologous compositionby: mixing blood from the mammal with a quantity of about 4% citric acidsolution; centrifuging the blood and citric acid mixture to separate aplatelet rich plasma component therefrom; and collecting the plateletrich plasma component; mixing the first component with the secondcomponent to provide the autologous composition; and administering theautologous composition to the mammal.